Cell manipulation techniques are important in many areas of research including cell biology, molecular genetics, biotechnological production, clinical diagnostics and therapeutics. Physical methods of manipulating suspended cells at single-particle microscopic resolution include hydrodynamic, optical, dielectrophoretic, magnetic and ultrasonic cell trapping.
Of the above-mentioned methods, ultrasound trapping has been less extensively exploited. Compared to other methods, ultrasonic cell manipulation is an inexpensive non-contact technique that allows simultaneous and synchronous manipulation of a large number of cells in a very short time. It is simple in both set-up and operation, and is non-invasive, chemically inert (non-toxic) and physically non-destructive. Taking into account its high efficiency and reliability and the fact that it can be used with the majority of cell types, this technique holds great promise in cell manipulation techniques for a variety of applications.
Forces acting on particles in an acoustic resonator have been studied under different conditions and for different goals. The origin of acoustic forces generated by ultrasonic standing waves has been extensively exploited. Depending on the experimental conditions, some of those forces can be generated and quantified. The most studied acoustic force is the PRF (primary radiation force), which is the force generated by an ultrasonic standing wave occurring between the walls of a resonator. In the case of a closed fluid-filled chamber, resonance is obtained when its thickness is equal to: w=Nλ/2 where N is the number of nodes in the chamber and λ is the ultrasound wavelength. The acoustic force Fac drives particles towards the nodes or antinodes depending on their acoustic properties. Fac=VkÃ<Eac>sin(2kx), where V is the particle volume, k=2π/λ is the wave number, Ã=[3(ρp−ρf/(2ρp+ρf)−(cp2ρp/cf2ρf)] is the acoustic contrast factor, where cp and cf are the sound speeds of the particles and the fluid respectively, and <Eac> is the average acoustic energy density. It is well known that the PRF appears as a second order of the Navier-Stokes equation. The order of magnitude of the acoustic force is really low (˜10−9 N) but high enough for influencing micron sized particles, cells, lipids or even bacteria.
A number of other primary and secondary acoustic forces have also been described, namely: the primary and secondary Bjerknes forces, the secondary interparticle force and the force generated by the transversal component of the PRF; the latter is considered to be the governing force influencing the aggregation process. Several reports have demonstrated that all of the forces mentioned above are at least two orders of magnitude smaller than the axial component of the primary force. Nevertheless, when particles reach the nodal plane the axial net force is zero, thus unveiling the transversal forces leading to the initiation of aggregation.
The aggregation process starts when isolated particles in levitation converge towards a specific point, where the acoustic energy is maximal.
The transversal migration of particles in the nodal plane is due to a complex coupling between forces generated by the non-homogeneity energy field distribution in the chamber and the transversal PRF originated by the imperfections of the resonator (for instance, the parallelism of the resonator walls and the lateral walls vibrations). The particle transversal force is therefore difficult to predict; nevertheless, by measuring the particle transversal velocity the mean transversal force FTr can be estimated, by considering that it is balanced by the Stokes' force Fs, such that: FTr−Fs=0, where Fs=3πηdνTr, with d being the particle diameter, η the dynamic viscosity and νTr the transversal velocity at the levitation plane. The complexity of the interactions of those forces may make the aggregation process difficult to control. It has been however reported that 2-D or 3-D aggregate configurations can be controlled by the initial concentration of cells.
It has been previously reported the use of an ultrasound standing wave trap (USWT) capable of holding >10,000 cells at the focal plane of a microscope. The USWT is an ultrasound resonator where the acoustic path-length in the cell suspension is a single half wavelength. The resonator has a pressure node plane half way through the cell suspension and parallel to the transducer. The cell trap exploits the fact that cells experience an axial direct acoustic radiation force when in an ultrasound standing wave field. This force drives them towards a node plane. They then move, within that plane, to accumulate at the centre of the field, i.e. at the nodal plane. The USWT has been used to synchronously and rapidly (within 10 s of seconds) form and levitate 2-D and 3-D cell aggregates in suspension away from the influence of solid substrata.
At low cell concentrations (≦5×105 cells/ml), 2-D aggregates may be generated, while at concentrations of ≧106 cells/ml, 3-D aggregates may be generated. This presents a relatively narrow particle/cell concentration margin over which 2-D aggregates can be formed. While, undoubtedly 3-D aggregates are more tissue-mimetic, there is still great interest in the 2-D form of the aggregate as this facilitates light microscope resolution of the interaction processes occurring between cells.
In addition, some of the known techniques may not allow a satisfactorily control aggregation mechanism. For example, one way of controlling the aggregation velocity is by tuning the resonance frequency. However, in this case, particle velocities change rapidly at resonance, resulting in poor reproducibility. A need thus exists to obtain a method allowing a more controlled aggregation mechanism.
Another need exists to obtain a method allowing the selective generation of 2-D or 3D aggregates.
The present invention aims to meet one or more of the aforementioned needs.